Vol. 69, No. 3, 1976

SOLUBILIZATION

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND PARTIAL

PURIFICATION

OF ALANINE CARRIER

FROM MEMBRANES OF A THERMOPHILIC BACTERIUM AND ITS RECONSTITUTION INTO FUNCTIONAL VESICLES Hajime

Hirata, Nobuhito Sone, Masasuke Yoshida and Yasuo Kagawa Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, Japan 329-04

Received

January

27,1976 SUMMARY

An alanine transport carrier was solubilized from membranes of the thermophilic bacterium PS3 with cholate-deoxycholate mixture. It was then partially purified by diethyl aminoethyl cellulose column chromatography and gel filtration. For assay of alanine carrier activity it was reconstituted into vesicles with P-lipids and the transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. The partially purified carrier had no ATPase or NADH dehydrogenase activity. Active transport of alanine driven by the membrane potential was completely abolished by an uncoupler.

Bacterial tensively

membrane transport

using

isolated

showed that

coupling

interpreted

by Mitchell's

obtain

bacterial riers

of metabolic

no transport

are too studies It

branes

unstable

various

the

drugs,

a dicyclohexyl from this

that

thermophilic organic

solvents

using

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

However,

to

required. from

known transport

be obtained

PS3, which

car-

Thus for

from the

are resistant (6).

ATPase complex

and noninonic 665

be

required.

and detergents

sensitive ionic

is

could

bacterium

carbodiimide

bacterium

this

(2 - 5).

from membranes.

material

results

been isolated all

in-

can best

system is

has yet

to be solubilized

seemed possible of

theory

may be because

a more stable

Recent

to transport

a much purer carrier

This

has been studied (1).

energy

chemiosmotic results

membranes.

further

Copyright All rights

membrane vesicles

more convincing

Unfortunately,

of nutrients

Indeed

mem-

to recently

was purified

detergents

(7).

Vol. 69, No. 3, 1976

This

BIOCHEMICAL

communication

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

describes

the

purification

of an alanine

carrier

constitution

with

into

port

P-lipids

solubilization

from this vesicles

and partial

bacterium

capable

of

and its

active

re-

trans-

of alanine.

MATERIALS AND METHODS Materia& The thermophilic bacterium PS3 (kindly donated by Dr. T. Oshima) and preparation of membranes were described previously (6). Soybean P-lipids (asolectin) were obtained from Associated Concentrates, Woodside, New York, and partially purified as described previously (7). The preparation of lipids of PS3 was was purchased also as described previously (7). L-[U- 14C]alanine from Daiichi Radiochemicals. Cholic acid was recrystallized as described by Kagawa (8). Other compounds were commercial products. P-Lipid-detergent Mixture P-Lipids (10 mg) were suspended in 2 ml of 40 mM Tris-SO4 (pH 8) containing 2% Na-cholate, 1% Na-deoxycholate and 1 mM dithiothreitol, and sonicated in a Tomy probe sonic oscillator, Model UR-150P, at 20 KHz and 150 watts in an ice bath for 5 min. Soihbilization and Partial Purification of Alanine Carrier Membranes (1 g of protein) were suspended in a solution containing Na-cholate, 2%; Na-deoxycholate, 1%; NapSOt,, 0.2 M; Tris-SOI, (pH 8), 30 mM; and dithiothreitol, 0.5 mM in a final volume of 100 ml. The mixture was sonicated in an ice bath for 10 min and then centrifuged at 140,000 x g for 1 hr. The supernatant was concentrated to approximately 20 ml in a Diaflo apparatus with a UMlO filter and dialyzed against 2 liters of 50 mM Tris-SOI, (pH 8) containing 0.25 mM dithiothreitol for 17 hrs at room temperature. The contents in the dialysis bag were then centrifuged at 140,000 x g for 1 hr and the resulting precipitate was suspended in 50 mM Tris-SO,+ (pH 8). This preparation is named CDE-P. CDE-P was treated with Triton X-100 (2% final concentration) in the presence of 0.2 M NapSOb, sonicated for 1 min and centrifuged at 140,000 x g for 1 hr. The supernatant was diluted 20-fold with distilled water and applied on a DEAE cellulose column equilibrated with 25 mM Tris-SO,, (pH 8) containing 0.1% Triton X-100 (Tris-Triton). The column was washed with 10 bed volume of Tris-Triton, and then eluted with Tris-Triton containing 50 mM NazS04. Fractions with UV-absorption were combined and mixed with Na-cholate at a final concentration of 1%. Then 210 mg of solid ammonium sulfate were added. The precipitate was collected by centrifugation (20,000 x g, 10 min) and suspended in 50 mM Tris-SOI, (pH 8) (DE-l). DE-1 was purified further by gel For this it was dissolved in a minimal volume of 2% filtration. Triton X-100 and applied on a Sepharose 6B column equilibrated with Tris-Triton. The column was eluted with the same buffer and the eluate with UV-absorption (one broad peak) was collected and divided into three fractions (Fr. 1, 2 and 3). Reconstitution of Vesicles and &-Loading Samples of the preparations obtained at each step (10 to 100 pg protein) were added to the P-lipid-detergent mixture (2 ml) described above and sonicated for Then the mixtures were dialyzed against 50 mM Tris-SOQ 30 sec. (pH 8) containing 2.5 mM MgSOs, 0.2 mM EDTA and 0.25 mM dithiothreiThe reconstituted vesicles were incuto1 for 17 to 20 hrs at 40". bated in 0.5 M K-phosphate buffer (pH 8) for 30 min at 55O and then

666

BIOCHEMICAL

Vol. 69, No. 3,1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

transferred to an ice bath. Then MgSO4 was added to a final concentration of 10 mM and incubation was continued for 30 min in the ice bath. The reconstituted-Kf-loaded vesicles were collected by centrifugation at 140,000 x g for 60 min, washed with 0.4 M sucrose containing 10 mM MgSO1, and suspended in 0.4 M sucrose-10 mM MqSO,+. Assay of ALanine Carrier Activity Alanine carrier was defined as protein(s) catalyzing active transport of radioactive alanine into the reconstituted vesicles depending on an artificial membrane potential (negative inside). The reconstituted-K+ -loaded vesicles were suspended in 1 ml of solution containing final concentrations of 0.1 M Tris-maleate, pH 7.0, 0.013 M MqSO4, 0.28 M sucrose, and 10 PM L-[U-14C]alanine, (1 vCi/ml). After incubation for 5 min at 400, 2 ug of valinomycin in 2 ~1 of methanol were added. At intervals 0.1 ml samples were filtered through membrane filters (Sartorius, 0.45 pm pore size) and washed with 0.4 M sucrose-10 mM MqS04. The filters were then rapidly removed, dried and their radioactivities were assayed in a gas-flow counter (Aloka LBC-451). Protein was determined as described by Lowry et al. (9).

RESULTS AND DISCUSSION A transport catalyzing

movement of

tration

gradient

simplified

in other

alanine

uptake

artificial

nutrient

carrier

a metabolic

form.

(manuscript

for

coli

assay of

of

energy.

similar In this

carrier

must be

work

PS3 was driven

by K+ diffusion

in preparation)

alanine

of

in a

energy

In our previous

introduced

(4).

a concen-

However,

protein(s),

membrane vesicles

vesicles

as protein

a membrane against

only

by isolated

Esckerickia

across sort

containing than

must be determined

on some

membrane potential

valinomycin

method

protein(s)

dependent

system

supplied

with

carrier

to that

mediated

after

by

achieved

work we applied

activity

by an

this

reconstitution

of vesicles. Figure vesicles

reconstituted

soluble tuted

1 illustrates

proteins. from

alanine from the

which

2.

On addition

reached

insoluble

on alanine

uptakes

from cholate-deoxycholate of valinomycin,

the detergent-soluble

The validity in Fig.

results

a maximum in

proteins of

The initial

proteins

this

of

alanine

667

vesicles

Vesicles active

assay method is rate

soluble

showed rapid

2 min.

showed less

by K+-loaded

reconstiuptake

of

reconstituted

uptake.

confirmed

uptake

and in-

by the

results

was proportional

to

Vol. 69, No. 3, 1976

BIOCHEMICAL

-2

AND BIOPHYSICAL

5

0 TIM

Fig.

Fig.

1

2

:

:

RESEARCH COMMUNICATIONS

7

(MIN)

+ Alanine uptake by reconstituted, K -loaded vesicles. Vesicles containing the detergent-soluble ( 0) or -insoluble ( 0) proteins were reconstituted and loaded The with K-phosphate (pH 8) as described in the text. reaction mixture (1 ml) contained final concentrations of 0.1 M Tris-maleate, pH 7.0, 0.013 M MgS04, 0.28 M and 10 UM L-[U-14C]alanine, (1 uCi/ml). After sucrose, incubation for 5 min at 40" (arrow) 2 pg of valinomycin in 2 ~1 of methanol were added. At intervals, 0.1 ml aliquots were filtered and washed.

Alanine uptake by reconstituted-K+-loaded besicles as a function of amount of detergent-soluble proteins added. Reconstituted, K+-loaded vesicles containing various amounts of detergent-soluble proteins and a fixed amount of PS3 P-lipids (10 mg) were prepared as described in the text. The initial velocities ( 0) and maximal uptakes (0) were calculated from the values observed 15 set and 2.5 min after addition of valinomycin.

668

Vol. 69, No. 3, 1976

the

BIOCHEMICAL

amount of protein

ed to a fixed

(CDE-P),

hand,

explained

by supposing

tended

to both

carrier

over

amount of P-lipids

the other

tional

AND BIOPHYSICAL

the

protein(s)

the

range

to reach

a definite

that

initial

size

of the

and the

rate

population

level

was related

thus

reached

a definite

level

when most of

by these

on an artificial blocked

containing in the vesi-

former

only

the vesicles

and

contain

at

protein(s).

The uptake

ly

be

was propor-

protein(s) to the

on

could

of uptake

amount of carrier

the maximal

level,

This

of vesicles

while

one carrier

20 to 600 i-19, add-

level.

cles,

least

of

The maximal

(10 mg).

the

RESEARCH COMMUNICATIONS

reconstituted

vesicles,

membrane potential

by the uncoupler,

phenyl-hydrazone,

but

(negative

which inside),

carbonylcyanide

was dependent was complete-

p-trifluoromethoxy-

not by dicyclohexyl

carbodiimide

(data

not

shown). Various P-lipids, rial

P-lipid

soy bean P-lipids,

strain

phosphatidyl

with

alanine

Thus specific of the

carrier

phosphatidyl

saturated

fatty

only

are required

acyl

groups

not

vesishown).

reconstitution

of PS3 P-lipids

glycerol

of which

formed (data

functional

phosphatidyl

bacte-

and synthetic

activity

for

PS3

thermophilic

PS3 P-lipids

The main components

ethanolamine,

and the

(111,

from other

showing uptake

protein.

including

HB8, ATCC 27634,

glycerol;

carrier

P-lipids

were tested,

those

Thermus thermophilus

dipalmitoyl cles

preparations

are

and cardiolipin

are almost

completely

(7).

The alanine

was purified

as described

in the

AND METHODS" and results

of

purification

are summarized

in Table

of extraction

I.

carrier

The process

cholate-deoxycholate 2.4-fold. lulose

mixture

The alanine column with

carrier

rather

a typical

increased activity

a low salt

669

"MATERIALS

of the membranes with the

specific

was eluted concentration,

activity from a DEAE celsometimes

as

low

Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Purification

of Alanine

Membrane vesicles Cholate-DOC ext. Triton

ext.

Triton DEAE

SP. Act. n moles/min*mg

1,096 96.3

0.76 1.85

sup

DEAE 200 mM Na2S04 Sepharose Fr. 1

as 25 mM Na2S04. but

small

tion

alanine

specific

step

procedures

16.0 0.65

0.77 9.93

0.64 0.18

5.88 1.84

were still

1 of Table

I.

must be between

of the

it

0.50 5.12

final

contained

are required

present

On Sepharose

membranes and the that

1.89

37.8 2.5

removed ATPase and NADH dehydrogenase

shown).

in Fr.

carrier

activity

original

suggesting tive

not

was found

of the

the

(data

49.7

2 3

amounts of cytochromes

eluted

activity

This

Fr. Fr.

Carrier

Protein hg)

ext. ppt 50 mM Na2S04

Sepharose Sepharose

I

in the prepara-

6B column,

Thus the molecular 100,000

was about

preparation

was

if

the

The

13-fold

that

of

colorless,

almost

any cytochrome.

to increase

weight

and 200,000.

preparation

little

the highest

More effec-

yield.

ACKNOWLEDGEMENTS The authors Science

Institute,

bacterial

strain

Ltd.

for

sistance

large

are grateful Machida used. scale

of Misses

to Dr. City,

They also culture

Toshiko

Tairo

Tokyo, thank

Oshima of Mitsubishi

for

kindly

Keiko

is acknowledged.

670

the

members of Kyowa Hakko Co.,

of the bacterium. Kanbe,

providing

Life

Ikeba

The technical and Machiko

as-

Yoshimura

Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Kaback, H. R. (1974) Science 186, 882-892 Mitchell, P. (1966) Biol. Rev.41, 445-502 Harold, F. M. (1974) Ann. N. Y. Acad. Sci. 227, 297-311 Hirata, H., Altendorf, K., and Harold, F. M. (1974) J. Biol. Chem. 249, 2939-2945 Altendorf, K., Hirata, H., and Harold, F. M. (1975) J. Biol. Chem. 250, 1405-1412 Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1975) J. Biol. Chem. 250, 7910-7916 Sone, N., Yoshida, M., Hirata, H., and Kagawa, Y. (1975) J. Biol. Chem. 250, 7917-7923 Kagawa, Y., and Racker, E. (1971) J. Biol. Chem. 246, 5477-5487 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Oshima, T., and Imahori, K. (1974) International J. of Systematic Bacteriology 24, 102-112 Kagawa, Y., Sone, N.,%irata, H., Yoshida, M., and Okamoto, H. in preparation

671

Solubilization and partial purification of alanine carrier from membranes of a thermophilic bacterium and its reconstitution into functional vesicles.

Vol. 69, No. 3, 1976 SOLUBILIZATION BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS AND PARTIAL PURIFICATION OF ALANINE CARRIER FROM MEMBR...
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